CN102204512A - Tissue culture method for lilium tenuifolium - Google Patents
Tissue culture method for lilium tenuifolium Download PDFInfo
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Abstract
The invention discloses a tissue culture method for lilium tenuifolium, which comprises: sterilizing stripped lilium tenuifolium bulb serving as an explant from an intermediate scale part; and inoculating the stripped lilium tenuifolium bulb into a Murashige and Skoog (MS) medium, a first-generation induction medium consisting of 6-benzyladenine (BA) at a concentration of 1.0mg/L, nicotinic acid amide (NAA) at a concentration of 0.1mg/L and vitamin C (Vc) at a concentration of 5mg/L, a sub-generation propagation medium consisting of MS, 6-BA at a concentration of 0.2mg/L and NAA at a concentration of 0.01mg/L, a rooting medium consisting of 1/2of MS, and indole-3-butyric acid (IBA) at a concentration of 0.5 mg/L and bulbing culture medium consisting of 1/2of MS, IBA at a concentration of 0.5 mg/L and 2 percent of white sugar in turn for culture under the culture conditions including a culture temperature of 25+/-1 DEG C, day light and 4 hours of night light supply per day. When the culture method is used, tissue culture lilium tenuifolium seedlings propagated indoor in large scale can be obtained in a short time period, the productivity is high and the potential ecological benefit and social benefit can be created.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, relate to a kind of tissue culture of Lilium tenuifolium and the foundation of regenerating system, comprise the selection and the sterilization of explant, the screening of different times medium, the influence that condition of culture grows to culture, the contents such as foundation of quick propagating technology and regenerating system.
Background technology
Lilium tenuifolium (Lilium pumilum) has another name called morningstar lily flower, is the perennial bulb class of Liliaceae lilium flowers.The high 30-40 of strain stalk centimetre, the stem acropetal leaf, elongated slim and frahile, long and narrow as pine needle.Spend saggingly, the end of spring and the beginning of summer is open, the outside warp of petal, and look scarlet, immaculate usually, near sometimes base portion has the minority spot, and is glossy, and tool delicate fragrance is very beautiful.The florescence 6-8 month, the fruit phase 8-9 month.Give birth to patana or border.Be distributed in provinces and regions such as China Dark Longjiang, Jilin, Liaoning, Hebei, Henan, Shandong, Shanxi, the Inner Mongol, Shaanxi, Ningxia, Gansu, Qinghai.
Lilium tenuifolium strain shape is beautiful, is China northeast, North China, the famous wild flowers in the Northwest, should plant in flakes under sparse woods, in the lawn.And because its natural disposition is strong, it is drought-enduring to resist cold, under the condition of north arid, semiarid zone natural precipitation, get final product normal growth, be a kind of that distribution is the widest in the lilium, latitude is by north, meet very much the dry area in north china urban landscaping, beautify the needs of development, application prospect is very wide.But because its natural propagation coefficient is very low, and exists the nature degenerate problem, artificially excessively excavate the influence with natural cause in addition, make it become rare endangered plants.The method of at present common artificial propagation Lilium tenuifolium mainly is to adopt to divide to plant clove, seeding method and scale cuttage, not only breeds limited amount, also very easily causes disease to infect and quality deterioration.
Improved seeds lily are bred fast, detoxification rejuvenation, rearing new variety and Applied Biotechnology are carried out in the molecular breeding, and tissue culture all is relatively more suitable method.The tissue culture method breeding is drawn materials conveniently; be not subjected to the restriction of natural conditions; can constantly breed in a large number; it is the most effectual way of Lilium tenuifolium introducing and planting, quick breeding, detoxification rejuvenation and rearing new variety; can obtain a large amount of seedlings in a short time; solved the less problem of provenance in the cultivation, for Lilium tenuifolium research and scale, industrialization development utilization from now on provides theoretical foundation.Therefore, carrying out the Lilium tenuifolium Study on tissue culture in a deep going way has become researcher and has demanded one of work of carrying out urgently.
The tissue culture of lily starts from the 1950's, from nineteen fifty-seven Robb carries out the tissue culture achieving success first with lily bud scale after, lily in-vitro propagate technology is increasingly mature, and is very fast in the development of the aspects such as quick breeding, rearing new variety and germ plasm resource preservation of improved seeds.Up to now, the lily kind of tissue culture success both at home and abroad and kind have surpassed hundreds of, but what be more common in report is to be used for the cultivar that fresh cut-flowers is produced, and be less to the Study on tissue culture of the wild lily of China.Sum up the data in literature over nearly 20 years, the only wild lily tissue culture success of kind surplus in the of ten is about caning be counted on one's fingers especially of Lilium tenuifolium.
CN1460409 (03135142.5) discloses a kind of method for quickly breeding of lanzhou lily, choose the lanzhou lily scale, the segment of leaf and root, adopt explant sterilization back to insert the medium that contains different plant hormones, induce the generation bud, move into root media behind the successive transfer culture, be transplanted into plantation and practice seedling matrix in the time of the about 3-5 of test tube height of seedling centimetre, induce and the fast breeding culture medium of scale indefinite bud are MS, BA 2mg/L, NAA 0.2mg/L, root, leaf induce with propagating culture medium be MS, BA 2mg/L, NAA0.4mg/L, rooting of vitro seedling medium are 1/2MS, NAA 0.3mg/L, 27 ± 2 ℃ of temperature, intensity of illumination 2000Lx, light application time 12hour/day, pH are 5.8.CN101156552 (200710066339.1) discloses a kind of method of oriental lily tissue culture detoxicating, induce indefinite bud after selecting healthy lily bud scale to cut, indefinite bud is transferred on the growth medium, alternating temperature was cultivated after 30 days, cut stem apex and be transplanted in the growth medium and continue alternating temperature and cultivate after 30～60 days, just can obtain required avirulent strain system.Detoxification efficiency of the present invention is remarkable, the efficient height, and general kind can reach 90%～100%.It is easy and simple to handle, and is with low cost, has a good application prospect.
But above-mentioned medium and method also are not suitable for Lilium tenuifolium, and the present invention has systematically studied the optimal medium of different phase in the training of Lilium tenuifolium group, the suitableeest condition of culture and training method; Utilize the test tube clove to peel off to expand numerous and seed regeneration cultivation aspect have unique innovation; For the foundation of Lilium tenuifolium regenerating system provides science and technology support, solved the slow difficult problem of Lilium tenuifolium reproduction speed, for realizing that drought resisting landscape planting new concept has increased kind and had stronger practicality, for reliable basis has been established in the batch production production that realizes the Lilium tenuifolium bulb.
Summary of the invention
The objective of the invention is to utilize method for plant tissue culture; obtain the Lilium tenuifolium tissue cultivating seedling of indoor large-scale breeding in short time; with going to the factory of formation, scale, industrialization production; and it is low to overcome traditional bulb breeding rate; speed is slow; easily disease infects and the shortcoming of quality deterioration, and the new propagation method of tissue cultivating seedling can be provided throughout the year.
The scale that the present invention has chosen Lilium tenuifolium is an explant, and research comprises the selection of explant and sterilization, the screening of different times medium, the influence that condition of culture grows to culture,, the contents such as foundation of quick propagating technology and regenerating system.
In embodiments of the invention, comprise the method for tissue culture of a kind of Lilium tenuifolium (Lilium pumilum), it is characterized in that it is made up of following steps:
Step 1: the bulb that adopts Lilium tenuifolium carries out sterilization treatment as explant to it, is used for inducing of indefinite bud; Utilize test tube regeneration clove as explant again, directly insert and induce.
Step 2: explant is inoculated in MS+6-BA 1.0mg/L+NAA 0.1mg/L+V
c5mg/L's is first on the inducing culture, induces Lilium tenuifolium scale indefinite bud;
Step 3: the scale indefinite bud that is about 1-3cm that step 2 is differentiated takes out, and is inoculated on the shoot proliferation medium of MS+6-BA 0.2mg/L+NAA 0.01mg/L, breeds a large amount of Lilium tenuifolium tissue cultivating seedling;
Step 4: the Lilium tenuifolium tissue cultivating seedling that is about 3-4cm with step 3 differentiates, be inoculated on the root media of 1/2MS+IBA 0.5mg/L, directly carry out root induction;
Step 5: the Lilium tenuifolium that step 4 the is differentiated seedling of taking root, be inoculated in the balling medium of the edible white sugar 2% of 1/2MS+IBA 0.5mg/L+, induce and carry out balling.
The condition of culture of each step is: 25 ± 1 ℃ of cultivation temperature, and intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.
In embodiment of the present invention, in scope as well known to those skilled in the art, NAA, 6-BA, IBA, V
cBe respectively methyl, 6-benzyl aminopurine, indolebutyric acid, ascorbic abbreviation.
In embodiments of the invention, further according to the different parts of Lilium tenuifolium bulb induce the differentiation rate difference, show that by experiment scale differentiation capability (differentiation frequency and speed) is outside＞inside, inductivity is followed successively by skin＞middle level＞internal layer.And same scale different parts induces differentiation rate also to have notable difference, and inductivity is bottom＞middle part＞top in proper order, and promptly scale base portion differentiation capability is the strongest, takes second place in the middle part, and top is the poorest.Owing to outer scale mechanical damage degree height, the damage by disease and insect influence is serious, scale bacteria containing amount height again, the inoculation after stain is serious, and internal layer scale edge easily forms callus, the seedling growing way of acquisition a little less than, the growing state of the bud induction rate of middle level scale and inoculation back bud is all best, and pollution rate is low.In addition, when stripping and slicing less than 1cm
2The time produce differentiation hardly.
Therefore, the best explant when the present invention clearly proposes Lilium tenuifolium and carries out tissue culture is the middle level scale, and the growth potential of its inductivity and inoculation back bud is best, is that just generation is carried out the best explant of bud inducing culture.Also propose the scale vertically cut apart, scale is cut into stick, make every all to have base portion tissue, all can induce to break up for every and sprout, its inductivity all can reach more than 90%, and the differentiation rate of inducing of every scale has been increased exponentially.
In embodiment of the present invention, utilize test tube regeneration clove to be one and effectively improve the means that initial culture is induced differentiation rate.The regeneration inductivity of test tube clove can reach more than 80%, and pollution rate is almost nil, and by the differentiation capability of the scale of the test tube clove that obtains after the training of scale group originally＞originally the cultivate differentiation capability of scale.Like this, both having saved wild resource, and simplified the sterilizing program of explant again, and improved and induced differentiation rate, is the main method of accelerating breeding.
Simultaneously, in embodiment of the present invention, the inductivity that inoculation is inserted during Lilium tenuifolium behind the medium by different way is also different: the scale back side insertions＞base portion oblique cutting＞outside of belly insertion, the laying method of when operation inoculation scale is described and also influences its inducibility, and the easier induced bud of adaxial and its surface produces with the contact site of medium.
The present invention has also comprised the selection of agent of explant surface disinfection and sterilizing methods, to the variety classes disinfectant, the variable concentrations of identical type disinfectant, different disposal time and method compare, changed the conventional processing method that the scale explant is washed in flowing water, control moisture is noted in proposition in sampling and entire process process, successful control just for the pollution problem in inducing culture stage, greatly reduce pollution rate, improved and induced success rate.In the pre-treatment process, reduce the flushing and the soak time of running water as far as possible, except that washing a little, outer scale removes the earth, in, the internal layer scale generally do not need the flushing, the conventional on year-on-year basis situation of the flushing of scale explant and each processing procedure all being used sterile water drip washing under discharge condition is about pollution rate decline 30%-40%.
The present invention also passes through comparative test, filters out the Lilium tenuifolium group and trains each stage optimal medium:
A. just for inducing culture: MS+6-BA 1.0mg/L+NAA 0.1mg/+V
c5mg/L is the first for inducing culture of Lilium tenuifolium scale the best, and the newly-increased bud number of not only inductivity height, and each explant is many, and differentiation rate is the highest, and its differentiation rate can reach 80%.
B. shoot proliferation medium: MS+6-BA 0.2mg/L+NAA 0.01mg/L, the 6-BA of high concentration has inhibitory action, excessive concentration to lily bulb propagation, can suppress the growth of bud, along with increasing of 6-BA concentration, when surpassing 2.0mg/L, grow thickly and downgrade obviously, easily produce the vitrifying seedling.
C. root media: 1/2MS+IBA 0.5mg/L, low salt concn is favourable to taking root of Lilium tenuifolium, and rooting rate reaches 88.9%.Base portion can increase the clove of band root newly when taking root, and on average can reach 3-4.
D. balling medium: 1/2MS+IBA 0.5mg/L+ white sugar 2%, no matter Lilium tenuifolium is at MS or in the 1/2MS medium, sugar content increases all has facilitation to balling, the 1/2MS+IBA 0.5mg/L+ white sugar 2% of comparing is even more ideal, balling quantity is many, bulb is bigger, obvolvent degree height, and the emergence rate effect is all best peeling off and transplanting.
Also comprise acclimatization and transplants and hot-house culture stage after Lilium tenuifolium is taken root in the specific embodiments of the present invention: will be in root media robust growth, the long test-tube plantlet to 1-2cm of root carries out hardening and moves.At first carry out 2 days bottle hardening of closing, carry out 2-3 days uncork hardening again, change the greenhouse then over to and plant.Pour out test-tube plantlet during transplanting carefully, with flushing with clean water root residual medium, be stained with root 8min with the IBA of 50ppm after, plant in 4 kinds of matrix of prior design.The result shows that vermiculite+sheep excrement+diammonium phosphate is fit to the growth of seedling root system as the cultivation matrix best results very much, and transplanting survival rate can reach more than 80%.After tissue cultivating seedling grows to 3-4 sheet true leaf, can move into and heel in that the garden is heeled in or directly field planting, can execute the compound rich water of some dilutions period in right amount at this section.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
Embodiment 1. Lilium tenuifolium explant selection and method of operating are to the influence of adventitious bud inducing
1. materials and methods: explant is the bulb of Lilium tenuifolium, plants ball and picks up from periphery mountain area, Huhehaote City, the Inner Mongol, and acquisition time is will sprout spring or just sprout the time, and explant is taken from the scale position, middle level of Lilium tenuifolium.Subsequently explant is sterilized, treatment step is: scale wash a little remove earth → put on the super-clean bench sterile chamber → every liter drip detergent solution that 2-3 drips " Tween-20 " soak 60 seconds → change over to 75% alcohol soak 30 seconds → again with 0.15% 3-4 time → aseptic filter paper of mercuric chloride solution processing 20 minutes → sterile water drip washing on suck dry moisture → directly insert prepared culture medium in advance.The material that is noted that each processing is too much unsuitable, wants the continuous jog and the scale tissue of avoiding damaging in the whole process.Facts have proved that this Disinfection Effect is better, the effect of employing mercuric chloride solution also is better than the liquor natrii hypochloritis of common usefulness.Utilize test tube regeneration clove, regeneration back inductivity height on average can reach more than 80%.
2. the explant size is to the influence of Lilium tenuifolium scale survival rate and adventitious bud inducing
Scale is divided into five groups, is cut into the strip and the full wafer of 0.5cm * 0.5cm, 1cm * 1cm, half sheet, rip cutting respectively, be inoculated in MS+6-BA 1.0mg/L+NAA 0.1mg/L+V thereafter
cOn the 5mg/L inducing culture, culture environment is: 25 ± 1 ℃ of cultivation temperature, and intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.Added up the inductivity of survival rate and indefinite bud respectively on the 35th day, the result is as shown in table 1.
Concrete statistical formula is:
Survival rate=(the explant sum of viable explant number/inoculation) * 100%
Just for bud induction rate=(the explant sum of the explant number/inoculation of sprouting) * 100%
Shoot proliferation multiple=(sum of the bud number of the growing thickly/access individual plant bud that induces) * 100%
Rooting rate=(the explant sum of the explant number/inoculation of differentiation root) * 100%
Table 1 explant size is to the influence of Lilium tenuifolium scale survival rate and adventitious bud inducing
3. scale position influence that the Lilium tenuifolium indefinite bud is induced surely
Scale is divided into three groups of skin, middle level and internal layers, is inoculated in MS+6-BA 1.0mg/L+NAA 0.1mg/L+V
cOn the 5mg/L inducing culture, culture environment is: 25 ± 1 ℃ of cultivation temperature, and intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.Added up the inductivity of indefinite bud respectively on the 30th day, the result is as shown in table 2.
Table 2 scale position is to the influence of Lilium tenuifolium adventitious bud inducing
The screening of embodiment 2. each stage optimal mediums of Lilium tenuifolium tissue culture
1. hormone concentration and proportioning thereof are to the influence of Lilium tenuifolium scale adventitious bud inducing
After scale inserted in the inducing culture, the scale through 3-7 days about 40% changed aubergine into, began to transfer to green again through 6-30 days and formed kick gradually, and through 15-45 days, it is green that about 90% scale changes.Change green back 30-55 days at scale base portion, middle part or top formation indefinite bud, but the situation of base portion formation indefinite bud is more, top produces indefinite bud hardly.
Scale inserts and contains on the MS medium of different plant hormones and proportioning, and culture environment is: 25 ± 1 ℃ of cultivation temperature, and intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.Added up the inductivity of indefinite bud respectively on the 40th day, the result is as shown in table 3, and in order to add the V of 5mg in every liter of the brown stain that prevents explant
C, reached desirable effect.
Table 3 hormone concentration and combination thereof are to the influence of Lilium tenuifolium scale adventitious bud inducing
2. hormone concentration is to the influence of Lilium tenuifolium indefinite bud shoot proliferation
The 1-3cm scale indefinite bud that is about that scale is differentiated takes out, and is inoculated on the MS medium of the 6-BA, the NAA that contain variable concentrations, and culture environment is: 25 ± 1 ℃ of cultivation temperature, and intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.Observed its propagation and growing state respectively on the 45th day, the result is as shown in table 4.
Table 4 hormone concentration and combination thereof are to the influence of Lilium tenuifolium adventitious bud proliferation
3. salinity is to the influence of Lilium tenuifolium culture of rootage
Be about 3-4cm Lilium tenuifolium tissue cultivating seedling with what differentiate, be inoculated in respectively on MS and the 1/2MS medium, additional IBA0.5mg/L, culture environment is: 25 ± 1 ℃ of cultivation temperature, intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.Directly carry out root induction and observed its situation of taking root on the 20th day, the result is as shown in table 5.
The influence that table 5 inorganic salt concentration is taken root to tissue cultivating seedling
4. the influence that sugared concentration is cultivated the Lilium tenuifolium balling
Through experiment, no matter the Lilium tenuifolium tissue cultivating seedling is at MS or in the 1/2MS medium, when sugar content increases, balling all there is facilitation, the 1/2MS+IBA 0.5mg/L+ white sugar 2% of comparing is even more ideal, and balling quantity is many, and bulb is bigger, obvolvent degree height, effect all is best when emerging peeling off once more and transplant.
Acclimatization and transplants and hot-house culture after embodiment 3. Lilium tenuifoliums are taken root
To in root media, robust growth, the long test-tube plantlet of root carry out acclimatization and transplants, at first carry out 2 days bottle hardening of closing, carry out 2-3 days uncork hardening again, and change the greenhouse then over to and plant to 1-2cm.Carefully pour out test-tube plantlet during transplanting, with the residual medium of the clean root of flushing with clean water, be stained with root 8min with the IBA of 50ppm after, plant in 4 kinds of matrix of prior design.The result shows that vermiculite+sheep excrement+diammonium phosphate is fit to the growth of seedling root system as the cultivation matrix best results very much, and transplanting survival rate can reach more than 80%.After tissue cultivating seedling grows to 3-4 sheet true leaf, can move into and heel in that the garden is heeled in or directly field planting, can execute the compound rich water of some dilutions period in right amount at this section.
Claims (6)
1. the method for tissue culture of a Lilium tenuifolium is characterized in that it is made up of following steps:
Step 1: the bulb that adopts Lilium tenuifolium carries out sterilization treatment as explant to it, is used for inducing of indefinite bud; Utilize test tube regeneration clove as explant again, directly insert and induce;
Step 2: explant is inoculated in MS+6-BA 1.0mg/L+NAA 0.1mg/L+V
c5mg/L's is first on the inducing culture, induces Lilium tenuifolium scale indefinite bud;
Step 3: the scale indefinite bud that is about 1-3cm that step 2 is differentiated takes out, and is inoculated on the shoot proliferation medium of MS+6-BA 0.2mg/L+NAA 0.01mg/L, breeds a large amount of Lilium tenuifolium tissue cultivating seedling;
Step 4: the Lilium tenuifolium tissue cultivating seedling that is about 3-4cm with step 3 differentiates, be inoculated on the root media of 1/2MS+IBA 0.5mg/L, directly carry out root induction;
Step 5: the Lilium tenuifolium that step 4 the is differentiated seedling of taking root, be inoculated in the balling medium of edible white sugar of 1/2MS+IBA 0.5mg/L+2%, induce balling.
2. the method for tissue culture of the described a kind of Lilium tenuifolium of claim 1, it is characterized in that: the optimal culture condition of each step is 25 ± 1 ℃ of cultivation temperature, intensity of illumination 2500Lx, except that the natural lighting on daytime, night, light filling was 4 hours/day.
3. the method for tissue culture of the described a kind of Lilium tenuifolium of claim 1, it is characterized in that: the explant of step 1 is taken from the scale position, middle level of Lilium tenuifolium, is cut into strip subsequently, is used for inducing differentiation to sprout.
4. the method for tissue culture of the described a kind of Lilium tenuifolium of claim 1, it is characterized in that, the sterilization treatment process of the explant of step 1 is: scale washes a little and removes earth, on super-clean bench, put into sterile chamber, dripping 2-3 at every liter drips in the detergent solution of " Tween-20 " and soaked 60 seconds, change in 75% the alcohol and soaked 30 seconds, handled 20 minutes with 0.15% mercuric chloride solution again, sterile water drip washing 3-4 time, suck dry moisture on the last aseptic filter paper directly inserts in advance in the prepared culture medium.
5. the method for tissue culture of the described a kind of Lilium tenuifolium of claim 1 is characterized in that: in step 2 seeded process, the Lilium tenuifolium explant adopts the scale back side to insert medium.
6. the method for tissue culture of the described a kind of Lilium tenuifolium of claim 1 is characterized in that: the seedling of taking root of the Lilium tenuifolium after step 4 root induction is transplanted, and the matrix of cultivation is made up of vermiculite, sheep excrement, diammonium phosphate.
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